The present invention is generally directed to compounds having mitogen-activated protein kinase modulating activity, and to methods of screening such compounds and to compositions employing the same. Modulation of mitogen-activated kinase activity has been associated with the treatment of a variety of medical diseases, conditions, and/or symptoms thereof in warm-blooded animals including humans.
Intercellular communication is essential for the development and normal function of all multicellular organisms. Cells communicate and respond to extracellular signals by utilizing various cellular mechanisms called signal transduction pathways. These pathways involve a series of molecular events culminating in the activation of effector mechanisms that result in specific cellular responses. Certain disturbances in signal transduction pathways are involved in the pathogenesis of various diseases, conditions and symptoms thereof including cancer, cardiovascular disease, inflammation, autoimmune diseases (e.g. rheumatoid arthritis), neurodegenerative diseases (e.g. Alzheimer""s disease), and other diseases including acquired immune deficiency syndrome (AIDS) and the like.
One of the important signal transduction pathways is the mitogen-activated protein (MAP) kinase pathway. This pathway is essential in cellular growth and differentiation. It is known that blockage of the MAP kinase pathway suppresses tumor growth. In this signal transduction cascade, sequential activation of kinases and subsequent protein phosphorylation lead to the activation of transcription factors at the DNA level.
The sequential activation of kinases (also known as the protein kinase cascades) is a common mechanism of signal transduction in many cellular processes. Mitogen-activated protein kinases are believed to include up to five levels of protein kinases that sequentially activate each other through the process of phosphorylation. Among several mechanisms for activating these cascades, one is believed to be initiated by a small GTP binding protein which transmits the signal to the protein kinases. The signal is then transmitted down the cascade by enzymes at the various levels. The existence of multiple levels in each of the MAP kinase cascades is believed essential for signal amplification, specificity and tight regulation of the transmitted signals.
Nicholas S. Duesbery et al., Nature Medicine, Vol. 5/7 (July, 1999) report that tumor cells proliferate and spread throughout the body in apparent disregard of normal environmental cues. The inhibition of the mitogen-activated protein kinase signal transduction pathway enables tumor cells to revertto a non-transformed phenotype and bypass the arrest of tumor growth in the body. It is also known that many oncogenes activate the mitogen-activated protein kinase signal transduction pathway and it is this inappropriate activation that mediates the transformed phenotype.
The pathophysiology of many diseases involves dysfunctional intercellular signaling. As indicated above, in cancer, for example, some oncogenes encode signal transduction pathway proteins involved in the regulation of self proliferation. Expression of these oncogenes may induce chronic activation that results in uncontrolled proliferation. Thus, tumor cells are effectively released from normal regulation and thereby proliferate, enabling the tumor to enlarge. In conditions manifesting unstable angina and myocardial infarction, platelets aggregate and participate in the occlusion of coronary arteries by responding to a variety of signals released by the rupture of an atherosclerotic plaque. In this particular case, platelets actually respond appropriately to signals they are receiving, but they do so in an inappropriate place.
In autbimmune disease, signaling molecules produced by the processing and presentation of self-antigens activate cells of the immune system, and the activated immune cells respond by eliminating the offensive stimulus. Cells in the body that express the self-antigen are targeted for destruction, and once the function of these normal cells is compromised, symptoms of the autoimmune disease become evident.
Since the pathogenesis of many diseases can be traced to a dysfunction in intercellular signaling, compounds which modulate the signal pathway can have utility in the treatment of a variety of such diseases.
Pheromones are a class of chemicals that are communicative between animals of the same species and elicit stereotypical behavior and endocrine responses. Although there is a wide variety and a large number of pheromone chemical structures, a single pheromone molecule has been shown to have biological effects in non-associated species such as a species of insects and elephants. Some chemical similarities exist between pheromones at different species.
The term xe2x80x9cinvertebrate pheromonexe2x80x9d (or xe2x80x9cpheromonexe2x80x9d) in the context of the present invention should be understood as encompassing any chemical compound isolated from any invertebrate species which is produced and discharged from glands and external ducts and functions by influencing other members of the same species in one of the ways known in the art, and which possesses mitogen-activating protein (MAP) kinase modulation activity. One example of an invertebrate order are insects, one of the predominant species of insects being Lepidoptera.
The terms xe2x80x9cpheromonexe2x80x9d or xe2x80x9cpheromone compoundxe2x80x9d encompasses the natural pheromones as well as synthetic compounds which display a similar MAP kinase modulating activity. Such synthetic compounds include various derivatives of the natural pheromones, as well as analogs thereof.
The pheromone communication system involves the release of specific chemicals from a pheromone producer (emitter), the transmission of these chemicals in the environment to a receiver, and the processing of the signals to mediate the appropriate behavioral responses in the receiver. Some pheromone compounds are believed to activate the vomeronasal organ (VNO) which resides interior to the main olfactory epithelium (MOE) in a blind-ended pouch within the septum of the nose. In both locations, the VNO and MOE neuroepithelial dendrites terminate in specialized cilia containing specific receptors that bind odorants. This binding initiates a cascade of enzymatic reactions that results in the production of messengers and the eventual depolarization of the cell membrane.
The signal transduction mechanisms through which pheromone compounds exert their effect involve various pathways which are commonly found in various other biological processes.
Applicants have investigated pheromone compounds as a class of chemicals that have potential modulating activity for the MAP kinase signal transduction pathway. The potential of active pheromone compounds to modulate (i.e. activate or inhibit) the MAP kinase signal transduction pathways provides compounds which may be used for the treatment of a wide variety of diseases, conditions, and symptoms thereof as mentioned above. For example, MAP kinase pathway inhibitors have been described 20 as effective in treating cancer and other proliferative diseases such as psoriasis and restenosis as disclosed in A. J. Bridges et al., U.S. Pat. No. 5,525,625. MAP kinase pathway inhibitors have also been described as being effective in abolishing resistance to myocardial infarction induced by heat stress in M. Joyeux et al., Cardiovasc. Drugs Ther., Vol.14/3, pp.337-343 (June, 2000). It is also known that MAP kinase pathway activators can desirably affect wound healing and tissue repair.
It would therefore be a significant advance in the art of treating diseases, conditions and symptoms thereof to identify pheromone compounds possessing MAP kinase modulating activity. It would also be a further advance in the art to identify pheromone compounds which have MAP kinase modulating activity because of their potential for use in treating a variety of diseases, conditions, and symptoms thereof including cancer, autoimmune disease, psoriasis, restenosis as well as wound healing and tissue repair resulting from physical causes including, but not limited to, medical treatment such as chemotherapy. It would be a further advance in the art if pheromone compounds having MAP kinase modulating activity could be effectively identified from a library of pheromone compounds.
The present invention is generally directed to the discovery that certain pheromone compounds possess mitogen-activated protein (MAP) kinase modulating activity. In one aspect of the present invention, there is provided a screening method for identifying compounds having MAP kinase modulating activity from a library of pheromone compounds. In a further aspect of the invention there is provided pharmaceutical compositions containing as an active agent at least one MAP kinase modulator. Such composition have potential for use in the treatment of a variety of diseases, conditions, and symptoms thereof.
In a first aspect of the present invention, there is a provided a method of screening compounds to obtain those having mitogen-activated protein (MAP) kinase modulating activity, comprising the steps of:
a) providing a biological model for screening of compounds having MAP kinase modulating activity, the model being predictive for MAP kinase modulating activity;
b) testing a group of invertebrate-derived non-peptide, non-steroid pheromone compounds in the biological model to determine the presence of MAP kinase modulating activity; and
c) selecting at least one of the pheromone compounds possessing MAP kinase modulating activity in the model to obtain at least one selected pheromone compound.
In another aspect of the present invention, there is a method of selecting compounds having MAP kinase modulating activity, comprising the steps of:
a) providing data relating to the three-dimensional (3D) structure of at least one pharmacophore of a compound known to possess MAP kinase modulating activity;
b) providing a library of compounds, the library comprising at least one pheromone compound selected from invertebrate-derived, non-peptide, and non-steroid pheromones; and
c) analyzing the 3D structure of one or more compounds of the library and selecting a compound having a domain with a 3D structure at least substantially similar to the 3D structure of the pharmacophore.
Optionally, the above selection method further comprises the step of testing a selected compound in a relevant biological model which is predictive of the desired MAP kinase modulating activity.
In a further aspect of the present invention, there is provided a pharmaceutical composition comprising an effective amount of at least one invertebrate derived, non-peptide, non-steroid pheromone compound or derivative thereof having mitogen-activated protein kinase modulating activity in combination with a pharmaceutically acceptable carrier. Methods of using the compositions to modulate mitogen-activated protein kinase signal transduction pathways is also encompassed by the present invention.
In accordance with the present invention it has been discovered that certain pheromone compounds, derivatives and analogs thereof (hereinafter xe2x80x9cpheromone compoundsxe2x80x9d) possess MAP kinase modulating activity and that such pheromone compounds are a source of pharmaceutical agents which may be used for preventing and/or treating diseases, conditions or symptoms thereof. Compounds of the present invention having potential pharmaceutical activity may be selected first on their ability to modulate MAP kinase activity in the associated signal transduction pathway. Such target compounds may be screened from a library of pheromone compounds to provide compounds having the desired pharmaceutical uses which involve MAP kinase activity. xe2x80x9cModulating activityxe2x80x9d is defined herein as activity which either activates, inhibits or otherwise varies the action of MAP kinase and/or the corresponding enzyme cascade along the MAP kinase signal transduction pathway. The selection of compounds which either increases or slows the action of MAP kinase and/or corresponding enzyme cascade along the MAP kinase signal transduction pathway, may be carried out using known assays in the art. Alternatively, it may also be possible, in accordance with the present invention, to separately screen compounds which increases or activates the MAP kinase activity in the associated signal transduction pathway, and to separately screen compounds which cause a slowing or inhibiting effect on the MAP kinase activity using a separate assay. In addition, compounds of the present invention may be selected directly which are useful in indications known to involve MAP kinase signal transduction pathway.
xe2x80x9cActivatorsxe2x80x9d as used herein include pheromone compounds which increase or activate MAP kinase activity and/or a corresponding enzyme cascade of the MAP kinase signal transduction pathway.
xe2x80x9cInhibitorsxe2x80x9d as used herein include pheromone compounds which slow or inhibit MAP kinase activity and/or the corresponding enzyme cascade of the MAP kinase signal transduction pathway.
The term xe2x80x9ctherapeutic activityxe2x80x9d is defined herein as any activity of a compound on specific target cells, tissue or organ, or activity which achieves a specific effect within a body including prevention and/or treatment of a disease, condition or symptom thereof, associated with or related to MAP kinase signal transduction pathway in warm-blooded animals including humans. The term xe2x80x9ctherapeutic activityxe2x80x9d is further defined as any activity which is manifested in relatively low levels or concentration of the active compound, which is a concentration which gives rise to a biological effect within the body, not through a general systemic effect, but rather through a specific effect on specific targets within the body especially those responsive to modulation of the MAP kinase signal transduction pathway.
When the compounds are first selected on the basis of their ability to modulate MAP kinase activity, the selection may be carried out using one or more assays known in the art.
A biological model suitable for screening pheromone compounds for MAP kinase modulation activity may be based on a model which is known and acceptable in the literature for screening of such compounds to select those which have the desired activity. In some indications, the acceptable models are in vitro models, e.g. models involving testing of the effect of the compound on a cell or tissue culture. In other indications, a relevant model having a predictive value is an in vivo model involving laboratory animals, which may include rodents such as rats, mice or rabbits; xenograft models, e.g. an immune-compromised mouse carrying human tissue; and higher order animals such as cats, dogs and even primates. Furthermore, the relevant model may at times be a combination of in vitro and in vivo models, or a combination of several in vitro and/or several in vivo models. In addition, at times the relevant model may also be a model of non-living material such as a model of isolated membranes, a variety of biochemical assays, and the like.
In a preferred screening method, as set forth in Example 1 herein, pheromone compounds with MAP kinase modulating activity are identified using Rat1 cells grown in cell culture dishes. Samples of the pheromone compound are added to respective cell culture dishes containing the Rat1 cells and incubated. A control group is established by treating a separate group of cell culture dishes containing Rat1 cells with the same solutions absent the pheromone compound. After incubation, the Rat1 cells are stimulated with epidermal growth factor to stimulate MAP kinase activation in the Rat1 cells. Upon completion of the incubation period, the Rat1 cells in each of the culture dishes are separately processed to obtain the corresponding cell extract containing proteins indicative of active kinase. The proteins are then separated using known electrophoresis techniques and prepared as a blot for the detection and measurement of active MAP kinase. The resulting blot is analyzed using densitometric techniques to measure the corresponding activity of the tested pheromone compound. The results obtained are compared to the results obtained for the control to measure modulation of the active MAP kinase initially induced by EGF.
A pheromone compound having the desired MAP kinase modulating activity may be selected on the basis of its three dimensional (3D) molecular structure. The term xe2x80x9cpharmacophorexe2x80x9d is used herein as the structural domain of a compound which is associated with or related to a desired pharmaceutical or biochemical activity. In this manner, the structure of compound having a known pharmaceutical activity may be used as a structural template for efficiently selecting compounds having potentially similar pharmaceutical activity. Once the compounds are selected based on their molecular structure, the selected compounds are tested in the relevant biological model as described above, to determine the presence of the desired pharmaceutical activity.
In accordance with the method described above, the following compounds have been observed to possess mitogen-activating protein kinase modulation activity. MAP kinase activators include cis-7-tetradecenal, cis-7-tetradecenol, cis-7-dodecenyl ester, heneicosene-11-one, cis-6-heneicosene-11-one, cis4-tridecenyl ester, cis4-tridecen-1 -yl ester, 2-heptanone, cis-7-tetradecenyl ester, trans-5-decenyl ester, cis-2-methyl-7-octadecene, cis-9-heneicosene, trans-2, cis-13-octadecadienal, 14-methyl-cis-8-hexadecenal, 2-methyl-3-butene-2-ol, trans-10-dodecenol, cis-9-tetradecenyl-formate, cis-9-tetradecenol, trans-3, cis-8, cis-11-tetradecatrienol, cis-7-tridecenol, cis-9-pentadecenol, and cis-9-undecenyl ester. Applicants have also discovered mitogen-activated protein kinase inhibitors, namely, trans-3, cis-7-tetradecadienyl ester and trans-3, cis-8-tetradecadienyl ester. The ester of the above-identified compounds may be any ester, preferably an acetate.
The above-identified compounds were screened in accordance with the methods described above and thereby demonstrated the desired MAP kinase inhibitory or activating activity.
The non-peptide, non-steroid invertebrate-derived pheromones of the invention may be isolated from an invertebrate by any one of the methods known in the art. Alternatively, the pheromone compound of the present invention may also be chemically synthesized such as disclosed in U.S. Pat. No. 5,728,376 incorporated herein by reference. Many of the invertebrate pheromone compounds including insect-derived pheromone compounds may be synthesized through the fatty acid synthesis pathway.
The invertebrate-derived pheromone compounds of the present invention have a molecular weight typically less than about 500 Daltons, and more typically less than about 300 Daltons.
Typically, the pheromone compound comprises a straight or branched hydrocarbon chain of variable length (typically having a length of from about 6 to 30 carbon atoms, preferably from about 7 carbon atoms to 23 carbon atoms, more preferably from about 9 carbon atoms to 21 carbon atoms). The hydrocarbon chain may comprise one or more double or triple bonds which may be located at any position in the chain, the double bonds being in the cis or trans configuration. Typically, the side chains in a branched main hydrocarbon chain may include alkyl groups, alkenyl groups, and/or alkynyl groups, each side chain comprising from one to five carbon atoms. The main hydrocarbon chain may also comprise or be linked to a cycloalkyl or cycloalkenyl group having from about 3 to 7 carbon atoms.
The main hydrocarbon chain may be substituted at any location of the chain by one or more functional groups including, for example, a hydroxyl, a ketone, an aldehyde, an epoxy group, a carboxylic acid, an ester, a heterocyclic group, and an aromatic group.
In addition, the hydrocarbon chain of the pheromone compound may also comprise one or more additional groups which may, for example, be selected from ketones, halides (such as for example, F, Cl, Br, I), acetate esters, amines, thiols, thioesters, short chain alkyls (such as for example, methyl, ethyl, propyl, butyl, pentyl).
All of the above modifications may be carried out by known techniques utilized by one of ordinary skill in the art.
In accordance with the invention compounds which comprise modified functional groups in which, for example, an oxygen atom is replaced by a sulfur atom, may also be used and fall within the meaning of the term xe2x80x9cpheromone compoundxe2x80x9d as used herein. Thus, for example, a hydroxyl may be replaced by a thiol, an ester or a thioester. Another example of a modification may be the replacement of a hydrogen atom by a halogen atom, such as, for example, a bromine atom. All of the above modifications may be carried out by known techniques utilized by one of ordinary skill in the art.
In accordance with the invention, derivatives or analogs of the invertebrate pheromone compound may also be used if they substantially maintain the MAP kinase modulating activity of the parent pheromone compound. The activity includes the desirable preventive or therapeutic activity of the pheromone compounds observed to modulate MAP kinase activity along the MAP kinase signal transduction pathway.
A xe2x80x9cderivative or analogxe2x80x9d of a pheromone compound as used herein includes any compound having the basic structure of the invertebrate pheromone in which one or more functional groups are modified, but which substantially maintains the desired activity of the unmodified pheromone. Such derivative or analog may include geometric isomers of the corresponding pheromone compound having the same or similar level of MAP kinase modulating activity.
A derivative or analog which xe2x80x9csubstantially maintainsxe2x80x9d the activity of the unmodified pheromone, includes those displaying MAP kinase modulating activity of a magnitude of at least 30%, preferably at least 50% of the activity of the unmodified pheromone. Where the end biological effect of the pheromone compound includes the prevention and/or treatment of a disease, condition or symptom, a derivative or analog of the pheromone compound may be regarded as substantially maintaining the MAP kinase modulating activity, if it achieves a similar preventive or therapeutic effect at a non-toxic concentration.
It will be understood that at times, the modification of a pheromone compound to form a derivative or analog thereof, may achieve the desired therapeutic activity at the same time may result in the loss or alteration of one or more inherent properties associated with the unmodified pheromone compound.
The invention further provides a pharmaceutical composition suitable for administration to a warm-blooded animal including humans, comprising as an active agent an effective amount of a pheromone compound as defined herein having a desired therapeutic activity associated with the modulation of MAP kinase activity, and a pharmaceutically acceptable carrier.
The term xe2x80x9ceffective amountxe2x80x9d as used herein includes an amount of the active pheromone compound which provides a desirable preventive or therapeutic effect in a warm-blooded animal including human afflicted with a disease, condition or symptom related to MAP kinase activity in the MAP kinase signal transduction pathway. It will be understood that the effective amount will depend on various factors such as, for example, the nature of the indication for which the agent is used, characteristics of the treated warm-blooded animal, and mode of administration and the like, and will be routinely determined by one of ordinary skill in the art.
The effective amount of the pheromone compound will typically be in the range of from about 0.001 to 200 mg/kg/day, preferably from about 0.01 to 20 mg/kg/day.
The pharmaceutical composition comprising at least one pheromone compound having MAP kinase modulating activity may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavors, etc.) according to techniques such as those known in the art of pharmaceutical formulation.
The present invention further provides a method of treating warm-blooded animals including humans afflicted with a disease, condition, or symptoms which may benefit from modulation of MAP kinase activity comprising administering to the warm-blooded animal an effective amount of a pheromone compound being selected from one or more suitable screening methods disclosed herein and those that may be known in the art.
For use in the methods of the present invention associated with the prevention and/or treatment of diseases, conditions, and symptoms thereof, the pheromone compound may be administered to a warm-blooded animal including humans by one of a variety of administration modes, including, but not limited to oral, intravenous, intramuscular, transdermal, subcutaneous, topical, sublingual, rectal means, by nasal application, and the like. In addition, the pheromone compound may be a volatile substance and thus has the advantage of being administered by inhalation, resulting, in many cases in a very rapid response to the active agent with relatively little, if any, side effects.
When the pheromone compound of the present invention is administered orally, it may be administered in the form of a tablet, a pill, a capsule (e.g. a gelatin capsule), a powder or a pellet. Where a liquid carrier is used, the oral preparation may be in the form of a syrup, emulsion, or soft gelatin capsule. Nasal administration may be by nasal insufflation or as an aerosol, and internal administration such as rectal administration may be, for example, by use of a suppository. For topical administration the pheromone compounds may be, for example, in the form of creams, ointments, lotions, solutions, gels or transdermal patches.
The pheromone compounds of the invention may typically be administered with a pharmaceutically acceptable carrier which does not interfere with the efficacy of the active pheromone compound. The carrier may be selected from a large number of carriers known in the art and the nature of the carrier will depend on the intended form of administration and indication for which the active agent is used.
Tablets, pills and capsules containing the pheromone compounds of the invention may also include conventional excipients such as lactose, starch, and magnesium stearate. Suppositories may include excipients such as waxes and glycerol. Injectable solutions may comprise saline, buffering agents, dextrose, water glycerol, ethanol and solvents such as propylene glycol, polyethylene glycol and ethanol. Such solutions may also comprise stabilizing agents and preservatives which are typically antimicrobial agents (such as chlorbutol, benzyl alcohol, sodium benzoate, ascorbic acid, phenol, and the like) and antioxidants (such as butylated hydroxy toluene, propyl gallate, sulfites, and the like). Enteric coatings, flavorings, and dyes and colorants may also be used.
At times, the pheromone compounds of the invention may be incorporated within a liposome prepared by any of the methods known in the art. In addition, the pheromone compounds may be encapsulated in inert polymerized particles such as, for example, nano particles, microspheres, microparticles, and the like known in the art.
The pheromone compounds of the invention may comprise a single active agent or alternatively two or more such active agents which may exert an enhanced effect.
According to the methods of administration as encompassed by the present invention, the pheromone compounds may be administered separately or, alternatively, in combination with various other treatments administered to the patient for the same or different disease, condition, or symptom thereof.
The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the examples which follow, and from the claims, that various changes, modifications, and variations can be made therein without departing from the spirit and scope of the invention.
The following examples are submitted for illustrative purposes only and are not intended to limit the invention as encompassed by the claims forming part of the application.